AB0127 BRONCHOALVEOLAR LAVAGE (BAL) FLUID AND SERUM FROM PATIENTS WITH SYSTEMIC SCLEROSIS WITH INTERSTITIAL LUNG DISEASE (SSc-ILD) PROMOTE A PRO-INFLAMMATORY GENE SIGNATURE IN HUMAN PRIMARY LUNG FIBROBLASTS
Authors: Y. Al-Adwi, J. Westra, A. J. Stel, H. van Goor, D. J. Mulder
Journal: Annals of the Rheumatic Diseases (2022)

Background While circulating cytokines are frequently investigated in SSc patients, they may also play a role in local tissue, such as the lungs. Although most SSc patients show limited systemic inflammation, some studies have demonstrated that BAL fluid obtained from patients with SSc contains elevated levels of pro-inflammatory and pro-fibrotic cytokines. However, the relevance of the local milieu of the bronchial tree on the development of ILD has not been studied. We hypothesized that the bronchial milieu as represented by BAL fluid will have pro-inflammatory and pro-fibrotic effects on lung fibroblasts as the major effector cells in lung fibrosis. We also anticipated a greater pro-inflammatory and pro-fibrotic effect of BAL fluid obtained from patients with ILD (SSc-ILD) compared to patients without ILD (SSc). Finally, we hypothesized that serum obtained from both SSc-ILD and SSc will have a similar and concordant effect on lung fibroblasts, due to the systemic nature of the disease. Objectives To show the differential effect of BAL fluid and serum obtained from SSc patients with and without ILD on mRNA expression of pro-inflammatory and pro-fibrotic markers in human primary lung fibroblasts. Methods Serum and BAL fluid were collected from 3 SSc-ILD and 3 without ILD who were all treatment-naïve. ILD diagnosis was based on HRCT and lung function tests. Normal human primary lung fibroblasts were cultured and treated with either BAL fluid (5%) or serum (0.5%) from all individual patients. No treatment (CTRL) or treatment with pooled serum from healthy controls were used as control. After 4h, fibroblasts were harvested in TRIzol. The mRNA expression levels of inflammatory markers (Interleukin-6: IL-6, Interferon gamma-induced protein 10: IP-10), fibrotic markers (Connective Tissue Growth Factor: CTGF, Transforming Growth Factor: TGF-β and Alpha- Smooth Muscle Actin: α-SMA) were assessed using RT-qPCR. Nonparametric Mann-Whitney U test was used for comparison between groups. Correlation between BAL and serum treated fibroblast were calculated for the expression of the markers using Pearson correlation coefficient method. Results Fibroblasts treated with either SSc or SSc-ILD BAL fluids showed a significantly higher mRNA expression of IL-6 compared to control (Figure 1 a). The same was observed for IP-10, except for SSc serum which was not significant (Figure 1 b). When comparing the effects of BAL fluids between SSc or SSc-ILD patients, the effect of SSc-ILD BAL fluid was strikingly more profound than observed with SSc on both IL-6 and IP-10 (Figure 1 a,b). Similar effects were seen when fibroblasts were treated with SSc serum, where serum from SSc-ILD resulted in significantly higher expression of IL-6 and IP-10 compared to SSc (Figure 1 a,b). The effect of serum and BAL on IL-6 gene expression were strongly and significantly correlated (r=0.9; P=0.015) while were weakly correlated regarding IP-10 expression (r=0.4; P=0.3) (not shown). The fibrotic markers TGF-β and α-SMA showed no difference in expression in BAL or serum-treated fibroblasts compared to controls (Figure 1 c,d). Only the fibroblasts treated with SSc-ILD serum showed a significant increase in mRNA expression of the early fibrosis marker CTGF when compared to control or SSc serum (Figure 1 e). Conclusion We showed a clear pro-inflammatory effect of BAL fluid obtained from patients with SSc on human fibroblasts as demonstrated by mRNA expression of IL-6 and IP-10. This pro-inflammatory effect was 5-10 times more profoundly observed in SSc patients with ILD compared to those without ILD. Similar effects were observed when fibroblasts were treated with serum obtained from the same SSc patients where the BAL fluid and serum of each patient seemed to provoke a concordant pro-inflammatory effect. Although further studies are warranted, our results underline the systemic nature of SSc and provide new insights into the role of and interaction between the local bronchial and systemic milieu in ILD development. Disclosure of Interests Yehya Al-Adwi: None declared, Johanna Westra: None declared, Alja J. Stel: None declared, Harry van Goor: None declared, Douwe J Mulder Grant/research support from: Dr. DJ Mulder as an employee of the UMCG received research grants from Sanofi Genzyme which were paid to the UMCG.